Molecular mechanism of wedelolactone inhibits high glucose-induced human retinal vascular endothelial cells injury through regulating miR-190 expression

To investigate the effects and molecular mechanisms of wedelolactone (WEL) on high glucose-induced injury of human retinal vascular endothelial cells (HRECs). The cell injury model was established by incubating HRECs with 30 mmol/L glucose for 24 hour. HRECs were divided into control (Con) group, high glucose (HG) group, HG + WEL-low dose (L) group, HG + WEL-medium dose (M), HG + WEL-high dose (H) group, HG + miR-NC group, HG + miR-190 group, HG + WEL + antimiR-NC group, HG + WEL + antimiR-190 group. The kit detects cellular reactive oxygen species (ROS), superoxide dismutase (SOD), and malondialdehyde (MDA) content; cell apoptosis was analyzed by flow cytometry; miR-190 expression was detected by real-time quantitative PCR (RT-qPCR). Compared with Con group, the levels of ROS and MDA in the HG group were significantly increased (P < .01), the SOD activity and the expression of miR-190 expression were significantly decreased (P < .05), and the apoptosis rate was significantly increased (P < .01). Compared with HG group, the levels of ROS and MDA in HG + WEL-L group, HG + WEL-M group and HG + WEL-H group were significantly decreased (P < .05), SOD activity and miR-190 expression were significantly increased (P < .05), and apoptosis rate was significantly reduced (P < .05). Compared with the HG + miR-NC group, the levels of ROS and MDA in HG + miR-190 group were significantly reduced (P < .01), SOD activity was significantly increased (P < .01), and apoptosis rate was significantly reduced (P < .05). Compared with the HG + WEL + antimiR-NC group, the ROS level and MDA content in the HG + WEL + antimiR-190 group were significantly increased (P < .05), SOD activity was significantly decreased (P < .05), and apoptosis rate was significantly increased (P < .05). Wedelolactone can attenuate high glucose-induced HRECs apoptosis and oxidative stress by up-regulating miR-190 expression.


Introduction
Diabetic retinopathy (DR) is one of the major complications of diabetes and is the leading cause of acquired blindness in working-age adults. [1]High blood glucose is a major triggering factor for the progression of DR, as it promotes oxidative stress and induces damage to human retinal endothelial cells (HRECs). [2]Finding effective treatments for DR is of utmost importance.
5] Previous studies have demonstrated that WEL extract can inhibit oxidative stress response and alleviate hydrogen peroxide-induced oxidative stress damage in vascular endothelial cells. [6,7]However, the protective effects and mechanisms of WEL on high glucose-induced damage in HRECs have not been reported.
MicroRNAs (miRNAs) are endogenous noncoding RNAs of approximately 20 to 23 nucleotides in length, and their altered expression is involved in key pathways such as oxidative stress, inflammation, retinal neurodegeneration, and autophagy in the pathogenesis of DR. [8] It has been found that miR-190 is downregulated in hydrogen peroxide-treated H9c2 cardiomyocytes, and overexpression of miR-190 can protect H9c2 cells from apoptosis and oxidative stress damage induced by hydrogen peroxide. [9]However, there is limited research on the protective effects of miR-190 on high glucose-induced damage in HRECs.
In this study, we aimed to investigate the effects of WEL extract on high glucose-induced damage in HRECs and explore the potential involvement of miR-190 as a mechanism.Our findings will provide experimental evidence for the use of WEL in the treatment of Dr

Materials
All experimental tests were conducted at the Research Laboratory of Ocean University of China, funded by the Key Discipline Fund.HRECs were purchased from the Cell Bank of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.WEL (batch number 111885-201804, purity 95.0%) was obtained from the China Institute for Food and Drug Control.Lipofectamine 2000 was purchased from Invitrogen Corporation (Waltham, USA).miR-190 mimics, antimiR-190, and their respective controls (miR-NC, antimiR-NC) were provided by Beijing Liuhe Huada Gene Company.Reactive oxygen species (ROS) detection kit, Annexin-V-FITC/ propidium iodide (PI) apoptosis detection kit, and ECL staining kit were purchased from Shanghai Biyun Tian Biological Company (Shanghai).Malondialdehyde (MDA) content detection kit and superoxide dismutase (SOD) activity detection kit were obtained from Beijing Solaibao Biological Company (Beijing).Cleaved-caspase3 rabbit polyclonal antibody (ab2302), cleaved-caspase9 rabbit polyclonal antibody (ab2324), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) rabbit polyclonal antibody (ab9485) were purchased from Abcam (Cambridge, USA).

Cell culture, transfection, and experimental groups.
HRECs were seeded in low-glucose DMEM medium containing 10% fetal bovine serum (glucose concentration of 5.5 mmol/L) and cultured in a 5% CO 2 , 37°C incubator.When the cells reached 80% confluency, they were digested with trypsin and passaged at a 1:3 ratio.Log-phase HRECs were seeded into 6-well plates at a density of 2 × 105 cells per well.According to the instructions of Lipofectamine 2000, HRECs were transfected with miR-190 mimics, miR-NC, antimiR-190, and antimiR-NC, respectively, in 50% confluent cells.The transfected cells were collected after 48 hours for subsequent experiments.
The ethical approval was not necessary, since the research primarily involves cell experiment.
2.2.2.Measurement of ROS levels in cells using a detection kit.DCFH-DA was diluted in serum-free culture medium at a 1:1000 ratio to achieve a final concentration of 10 μmol/L.After collection, HRECs were suspended in the diluted DCFH-DA solution at a cell concentration of 1 × 106 cells/ mL and incubated at 37°C with gentle inversion every 3 to 5 minutes for 20 minutes.The cells were then washed 3 times with serum-free culture medium to remove the excess DCFH-DA that did not enter the cells.The total fluorescence intensity of the cells was measured using a flow cytometer, with a positive control sample used to determine the level of ROS production.

Measurement of SOD activity and MDA content
in cells using detection kits.HRECs were collected and centrifuged, and the pellet was resuspended in an extraction buffer.The cells were then sonicated to disrupt the cells, and the supernatant was collected.The SOD activity and MDA content in HRECs were measured according to the instructions of the SOD activity detection kit and MDA content detection kit, respectively.

Detection of apoptosis rate using flow cytometry.
HRECs were adjusted to a concentration of 1 × 106 cells/mL using 1 × binding buffer.About 5 µL of Annexin-V-FITC and 5 µL of PI were added to 500 µL of cell suspension and stained in the dark for 20 minutes.The fluorescence signals were collected using a flow cytometer, and the apoptosis rate of the cells was analyzed using FlowJo 8.7.1 software.

Detection of cleaved-caspase3 and cleaved-caspase9 protein expression using western blot.
HRECs from each group were washed once with PBS and lysed in radioimmunoprecipitation assay buffer on ice.Forty micrograms of protein were loaded per lane, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred onto a polyvinylidene fluoride membrane.The membrane was incubated with blocking buffer at room temperature for 5 hours, followed by overnight incubation at 4°C with primary antibodies against cleaved-caspase3 (1:200), cleaved-caspase9 (1 µg/mL), and the internal control GAPDH (1:2500).The membrane was then incubated with the corresponding secondary antibodies at room temperature for 1 hour.Immunobands were detected using an ECL staining kit, and the relative grayscale values of the bands were determined using Image-pro Plus software.

Detection of miR-190 expression using real-time quantitative PCR (RT-qPCR).
Total RNA was extracted from HRECs using TRIzol reagent.About 2 µg of total RNA were reverse transcribed into cDNA using a miRNA reverse transcription kit.RT-qPCR was performed using miR-190 primers, U6 primers as internal reference, and a miRNA Table 1 The effects of wedelolactone on high glucose-induced oxidative stress in HRECs.(mean ± SD, n = 9).fluorescence quantitative kit.The upstream primer for miR-190 was 5ʹ-GGTCTTTGATGATGATTCTGG-3ʹ, the downstream primer was 5ʹ-CTAGGCACAGTATTGAAGGTT-3ʹ; the upstream primer for U6 was 5ʹ-GCGCGTCGTGAAGCGTTC-3ʹ, and the downstream primer was 5ʹ-GTGCAGGGTCCGAGGT-3ʹ.

Groups
The expression level of miR-190 was calculated using the 2 −△△Ct method.

Statistical analysis
Three replicate wells were set up for each group, and the experiments were independently repeated 3 times.The experimental data were expressed as mean ± standard deviation (mean ± SD) if they followed a normal distribution and had equal variances.Independent sample t test was used for comparisons between 2 groups, and one-way analysis of variance (ANOVA) followed by SNK-q test was used for comparisons among multiple groups.A P value of <.01 was considered statistically significant.

The effects of WEL on oxidative stress in high glucoseinduced HRECs
Compared to the control group (Con), the high glucose group (HG) showed a significant decrease in SOD activity (P < .01)and a significant increase in MDA content and ROS levels (P < .01).
Compared to the HG group, the HG + WEL-L, HG + WEL-M, and HG + WEL-H groups showed a significant increase in SOD activity (P < .01)and a significant decrease in MDA content and ROS levels (P < .01).There were statistically significant differences in SOD activity, ROS levels, and MDA content among the HG + WEL-L, HG + WEL-M, and HG + WEL-H groups (Table 1).

The effect of WEL on apoptosis in high glucoseinduced HRECs
Compared to the control group, the HG group showed a significant increase in apoptosis rate, cleaved-caspase3, and cleaved-caspase9 protein expression (P < .05).Compared to the HG group, the HG + WEL-L, HG + WEL-M, and HG + WEL-H groups showed a significant decrease in apoptosis rate, cleaved-caspase3, and cleaved-caspase9 protein expression (P < .05).There were statistically significant differences in apoptosis rate, cleaved-caspase3, and cleaved-caspase 9 protein expression among the HG + WEL-L, HG + WEL-M, and HG + WEL-H groups (Figure 1; Table 2).

The effect of WEL on miR-190 expression in high glucose-induced HRECs
Compared to the control group, the HG group showed a significant decrease in miR-190 expression in HRECs (P < .01).
Compared to the HG group, the HG + WEL-L, HG + WEL-M, and HG + WEL-H groups showed a significant increase in miR-190 expression in HRECs (P < .01).There were statistically significant differences in miR-190 expression among the HG + WEL-L, HG + WEL-M, and HG + WEL-H groups (Table 3).

The effect of miR-190 overexpression on oxidative stress in high glucose-induced HRECs
Compared to the HG + miR-NC group, the HG + miR-190 group showed a significant increase in miR-190 expression and SOD activity in HRECs (P < .01),and a significant decrease in ROS levels and MDA content (P < .01)(Table 4).

The effect of overexpression on apoptosis in high glucose-induced HRECs
Compared to the HG + miR-NC group, the HG + miR-190 group showed a significant decrease in apoptosis rate and protein expression of cleaved-caspase3 and cleaved-caspase9 in HRECs (P < .05)(Figure 2; Table 5).

Inhibition of miR-190 expression reversed the effect of WEL (40 μmol/L) on high glucose-induced damage in HRECs
Compared to the HG + WEL + antimiR-NC group, the HG + WEL + antimiR-190 group showed a significant decrease in miR-190 expression and SOD activity in HRECs (P < .01),and a significant increase in ROS levels, MDA content, apoptosis rate, and protein expression of cleaved-caspase3 and cleaved-caspase9 (P < .01)(Figure 3; Table 6).

Discussion
High blood glucose is a major factor leading to retinal damage in the development of DR.Multiple pieces of evidence suggest that excessive production of ROS is associated with high glucose-induced oxidative damage in HRECs.SOD is an antioxidant defense enzyme that directly scavenges ROS to maintain the redox homeostasis.In diabetes, the production of ROS exceeds the clearance capacity, leading to the generation of lipid peroxidation product malondialdehyde (MDA). [10,11]In this study, high glucose stimulation resulted in increased ROS levels, elevated MDA generation, and decreased SOD activity in HRECs, indicating that high glucose stimulation induces oxidative damage in HRECs.WEL has been shown to have broad cellular protective effects. [12]Zhu et al [13] demonstrated that WEL can enhance the antioxidant enzyme activity, reduce pro-inflammatory cytokine levels, inhibit lipid peroxidation reaction, and alleviate inflammation and oxidative damage in foot cells induced by amphotericin B. Results from Ding et al [14] indicated that WEL protects human bronchial epithelial cells from oxidative damage induced by cigarette smoke extract by increasing SOD, catalase (CAT), and glutathione (GSH) activity and reducing MDA content.Additionally, Lu et al [15] reported that WEL alleviates oxidative stress and inflammatory damage in liver tissues induced by carbon tetrachloride by enhancing SOD and GSH-Px activity and reducing the expression of TNF-α, IL-1β, and IL-6.This study demonstrated that WEL inhibits MDA and ROS formation induced by high glucose in a concentration-dependent manner and enhances SOD activity, indicating its protective effect against high glucoseinduced damage in HRECs.Oxidative stress is associated with increased apoptosis in HRECs under high glucose conditions.In this study, WEL significantly inhibited high glucose-induced apoptosis in HRECs, reduced the expression levels of apoptosis initiation factor cleaved-caspase9 and apoptosis execution factor cleaved-caspase3, further confirming the ability of WEL to inhibit high glucose-induced damage in HRECs.[18] Studies have reported that miR-190 expression is decreased in hepatocellular carcinoma, and upregulation of miR-190 can inhibit liver cancer cell proliferation and metastasis, making it a potential therapeutic target for liver cancer. [19]In a mouse model of Parkinson disease, upregulation of miR-190 can suppress the activation of microglia and inflammatory response, reducing neuronal damage. [18]Moreover, overexpression of miR-190 significantly reduces neurological scores, brain water content, infarct area, and neuronal apoptosis in rats with ischemia-reperfusion injury, protecting against brain ischemia-reperfusion damage. [17]In this study, high glucose stimulation resulted in downregulation of miR-190 expression, while WEL increased miR-190 expression  in a concentration-dependent manner, suggesting that the protective effect of WEL may be related to the upregulation of miR-190 expression.Functional analysis of miR-190 showed that overexpression of miR-190 can inhibit high glucoseinduced oxidative stress damage and cell apoptosis in HRECs, downregulate the protein expression of cleaved-caspase9 and cleaved-caspase3, similar to the protective effect of WEL against high glucose-induced HRECs damage.Furthermore, inhibition of miR-190 expression significantly attenuated the protective effect of WEL against high glucose-induced apoptosis and oxidative stress damage in HRECs, further indicating the protective role of WEL in high glucose-induced HRECs damage through upregulation of miR-190 expression.
In conclusion, WEL can counteract high glucose-induced damage in HRECs by inhibiting oxidative stress and apoptosis, and this mechanism is achieved through upregulation of miR-190 expression, providing preliminary insights into the protective mechanism of WEL in DR and providing experimental evidence for the development of WEL as a therapeutic agent for Dr

Figure 1 .
Figure 1.Effects of Wedelactone on apoptosis of HRECs induced by high glucose.HRECs.human retinal vascular endothelial cells.Con = control group, HG = high glucose group.

Figure 2 .
Figure 2. Effect of overexpression of miR-190 on apoptosis of HRECs induced by high glucose.(A) apoptosis-related protein expression, (B) flow diagram of apoptosis.HG = high glucose group.

Figure 3 .
Figure 3. Inhibition of miR-190 expression reversed the effect of wedelactone on the apoptosis of HRECs induced by high glucose.(A) apoptosis-related protein expression, (B) flow diagram of apoptosis.WEL = wedelolactone.

Table 4
The effect of miR-190 overexpression on oxidative stress in high glucose-induced HRECs (mean ± SD, n = 9).

Table 5
Effect of overexpression of miR-190 on apoptosis of HRECs induced by high glucose (mean ± SD, n = 9).

Table 2
Effects of wedelactone on apoptosis of HRECs induced by high glucose (mean ± SD, n = 9).